Improvement of plant varieties through transformation has become increasingly important for modern plant breeding. Various gene transfer technologies allow the incorporation of foreign DNA molecules into plants genomes. The expression of genes encoded by such foreign DNA molecules (transgenes, genes of interest) can potentially confer new beneficial characteristics to the plant like, for example, improved crop quality or yield. The expression of transgenes can also allow for the use of plants as bioorganic factories.
Most gene transfer systems, such as Agrobacterium-mediated transformation or bombardment with DNA-coated particles, stably integrate heterologous genes in the nuclear genome of the plant by means of non-homologous recombination. Plant engineering using these methods has several drawbacks. These methods produce a population of transformants varying in their transgene copy number and whose expression is often unpredictable because of position effect variations or possible gene silencing. Most transgenic plants produced by these methods contains within their nuclear genome non-desired vector sequences associated with the gene of interest. Furthermore, the threat of transgene contamination of wild relative plants from genetically modified plant is a major environmental concern. The risk of transgene escape from genetically altered crops to wild relatives predominantly arises through pollen dissemination.
Plastid gene transformation is an important alternative for the expression of heterologous genes in plants (reviewed by Bogorad, Trends Biotechnol. 18: 257–263, 2000 and Bock, J. Mol. Biol. 312: 425–438, 2001). Although plastid genomes are relatively small in size, 120 to 160 kb, they can easily accommodate several kilo bases of foreign DNA within them. Insertion of foreign DNA in the plastid genome mainly occurs via homologous recombination and a transgene can be site directed at a particular locus using suitable homologous flanking regions. One of the major advantages of plastid transformation is that it is possible to obtain very high transgene expression. The plastid genome (plastome) is highly polyploid so the transgene is expressed from multiple gene copies in the plastid. The polyploidy of the plastid genome is such that a mature leaf cell may contain over 10,000 copies of the plastome. Also contributing to the high level of the plastid transgene expression is absence of position effect and gene silencing. Another major advantage is that plastids from most crop plants are only maternally inherited and thus, the ecological risk of plastid transgene escape through pollen-mediated out crossing is minimized. The basic DNA delivery techniques for plastid transformation are either via particle bombardment of leaves or polyethylene glycol mediated DNA uptake in protoplasts. Plastid transformation via biolistics was initially achieved in the unicellular green alga Chlamydomonas reinhardtii (Boynton et al., Science 240: 1534–1537, 1988) and this approach, using selection for cis-acting antibiotic resistance loci (spectinomycin/streptomycin resistance) or complementation of non-photosynthetic mutant phenotypes, was extended to Nicotiana tabacum (Svab et al., Proc. Natl. Acad. Sci. USA 87: 8526–8530, 1990), Arabidopsis (Sikdar et al., Plant Cell Reports 18:20–24, 1991), Brassica napus (WO 00/39313), potato (Sidorov et al., The Plant Journal 19(2):209–216, 1999), petunia (WO 00/28014),tomato (Ruf et al., Nature Biotechnology 19: 870–875, 2001), oilseed rape (Hou et al., Transgenic Res. 12: 111–114, 2003) and Lesquerella Fendleri(Skaijinskaia et al., Transgenic Res. 12: 115–122, 2003). Plastid transformation of protoplasts from tobacco and the moss Physcomitrella patens has been attained using polyethylene glycol (PEG) mediated DNA uptake (O'Neill et al., Plant J. 3: 729–738, 1993; Koop et al., Planta 199: 193–201, 1996). More recently, micro-injection of DNA directly in plastids of marginal mesophyll cells of intact tobacco plant resulted in transient expression (Knoblauch et al., Nature Biotechnology 17: 906–909, 1999) but stable transformants using this technique have yet to be reported. Stable chloroplast transformation by biolistics was also reported for the Euglenophyte Eugena gracilis (Doetsch et al., Curr Genet. 39:49–60, 2001) and the unicellular red alga Porphyridium sp. (Lapidot et al., Plant Physiol. 129: 7–12, 2002), the dominant selectable marker used for latter consist of a mutant form of the gene encoding acetohydroxyacid synthase which confers tolerance to the herbicide sulfometuron methyl. As previously mentioned, chloroplast transformation consists of integrating a foreign DNA at a precise position in the plastid genome by homologous recombination. The plastid transformation vectors consist of cloned plastid DNA, homologous to the targeted region, which flanks a selectable marker gene which itself is linked to a gene or several genes of interest. After transformation, the transgene(s) and the selectable marker are inserted together as a block of heterologous sequence in the targeted locus of the plastid genome via homologous recombination between the vectors plastid sequence and the targeted locus. In order to obtain stably transformed homoplasmic plants, i.e. plants having the foreign DNA inserted into every plastome copy of the plant cell, several rounds of subculture on selective media are required. This process facilitates the segregation of transplastomic and untransformed plastids and results in the selection of homoplasmic cells with gene(s) of interest and the selectable marker stably integrated into the plastome, since these genes are linked together.
Most stable plastid transformation demonstrated to date has been based on selection using the antibiotic resistance gene aadA (as referenced above) or NPTII (Carrer et al., Mol Gen Genet 241:49–56, 1993), to obtain homoplasmic plants. These selectable markers confer a specific selection phenotype, the green pigmentation (U.S. Pat. No. 5,451,513), which allows to visually distinguish the green pigmented transplastomic cells from cells having wild-type plastids which are non pigmented under selection conditions.
Most plastid transformation methods rely on the use of a selectable marker that confers a non-lethal selection. These selectable markers also confer a specific selection phenotype, the green pigmentation (U.S. Pat. No. 5,451,513) which allows one to visually distinguish the green pigmented transplastomic cells from cells having wild-type plastids that are non-pigmented under selection conditions. For example, plants transformed with the bacterial aadA gene which confers resistance to spectinomycin and streptomycin grow normally in the presence of either one of these antibiotics whereas untransformed plants are bleached. Transformed plants can thus easily be identified using chlorophyll as a visual marker. There is a limited number of selectable markers available for plastid transformation and the most reliable ones, such as aadA or point mutations in the plastid 16S rDNA and rps12 genes, confer resistance to the same antibiotics, spectinomycin and/or streptomycin. Selectable markers conferring resistance to other antibiotics such as kanamycin were shown to be much less effective for plastid transformation.
There have been concerns on the antibiotic resistance genes in genetically modified (GM) crops because of the potential risks of horizontal gene transfer to micro organisms in the environment or to gut microbes. These potential risks may pose even greater concerns with transplastomic plants because of the prokaryotic characteristics of the plastid genome and the by far larger copy number of the transgene per cell. It is therefore highly desirable to obtain transplastomic plants without antibiotic resistance genes.
Another alternative are methods wherein the antibiotic resistance gene is removed after the transformation. Once plants are homoplasmic for the transgene, the presence of the selectable marker gene in the plastome is no longer required. Removal of the selectable marker from a transplastomic plant has been achieved by two different methods. The first method, initially developed for recycling of the selectable marker from a transformed C. reinhardtii plastome (Fischer et al., Mol. Gen. Genet. 251:373–80, 1996) and recently extended to Nicotiana tabacum (Iamtham and Day, Nat. Biotechnol. 18: 1172–1176, 2000), relies on the plastid homologous recombination system to delete the marker gene from the plastid genome. Homologous recombination between direct repeated sequences flanking the selectable marker results in the deletion of the DNA segment. This method requires several rounds of self- or backcrossing before transgenic plants without the selectable marker are obtained. The second method uses the Cre/lox recombination system (Hajdukiewicz et al., The Plant J. 27: 161–170, 2001; Corneille et al., The Plant J. 27: 171–179, 2001) to excise the DNA segment. In this method the Cre-protein mediates the excision of a DNA segment located between two lox sites in direct repeat orientation. In order to remove the selectable marker, the Cre gene is introduced into the nuclei of the transplastomic plants either through nuclear transformation or through crossing. Once the selectable marker gene is removed, the Cre gene is eliminated by genetic crossing. Albeit successful, removal of the selectable marker using either one of these methods have some disadvantages. Both methods are lengthy processes as multiple rounds of crossing may be required, and both methods leave an undesired residual heterologous DNA segment in the plastid genome that serves no purpose. Furthermore, unspecific recombination causing DNA deletions was observed with Cre/lox recombination system (Hajdukiewicz et al., 2001). Thus, there is a need to develop other methods to obtain transplastomic plants without the stable integration of an antibiotic selectable marker in the plastid genome.
The application of antibiotic resistance genes as the selectable marker for plastid transformation is also limited by the requirement of the green pigmentation as the selection phenotype. A selection based on green pigmentation needs to be carried out in the light, which may not be the optimal culture condition for some plant cell cultures. Furthermore, not all the tissue types exhibit the green pigmentation phenotype under normal culture conditions, such as most of the cell cultures from cereal crops. It is therefore desirable to develop a selection strategy for plastid transformation in which the selection is not dependent on the green pigmentation.
Certain non-antibiotic resistance marker genes, such as the protoporphyrinogen oxidase gene (U.S. Pat. No. 6,084,155), 5-enolpyruvylshikimate-3-phosphate synthase genes (Daniell et al., Nature Biotech 16:345–348, 1998; Ye et al., The Plant Journal 25(3):261–270, 2001), and the bar gene (Lutz et al., Plant Physiology 125:1585–1590, 2001), can be effectively expressed in transplastomic plants. Most recently, EPSPS and bar gene are also reported being used in segregation approach to obtain herbicide resistant transplastomic plants with exclusion of antibiotic gene (Ye et al., Plant Physiology 133:402–410, 2003).
Another alternative is using the spinach betaine aldehyde dehydrogenase as the selectable marker that confers resistance to the lethal compound betaine aldehyde (Daniell et al., Curr Genet 2001 39:109–116, 2001).The present invention provides methods for plastid transformation in a wide variety of plant species and algae. It addresses the problem of removal of the antibiotic resistance marker which is delivered to the plastids during transformation and provides selection methods for homoplasmic transplastomic plants wherein green pigmentation of the selected phenotype is not a prerequisite.